Xylitol, a five-carbon sugar alcohol, is used as a natural sweetener in the food and confectionary industry. It has an anticariogenic effect that inhibits the growth of the tooth-decaying bacterium Streptococcus mutans (see: Mäkinen, K. K., J. Appl. Nutr., 44:16-28 (1992)). Its sweetness level is equal to that of sucrose, and it can replace sucrose on a weight-to-weight basis. When dissolved in water, xylitol has low viscosity and negative heat effects, and it does not require insulin for metabolic regulation. Owing to these benefits, the use of xylitol in the food industry is growing rapidly.
On a large-scale, xylitol is currently produced by chemical reduction of D-xylose derived mainly from wood hydrolysates. D-Xylose is a major pentose sugar found in lignocellulose and the second most abundant natural sugar (see: Ladisch, M. R., et al., Enzyme Microb. Technol., 5:82-102 (1983)). The conventional process of xylitol production includes four steps: acid hydrolysis of plant material, purification of the hydrolysate to pure D-xylose, hydrogenation of the D-xylose to xylitol, and crystallization of the xylitol (see: Aminoff, C., et al., In Counsell, J. N. (ed.). Xylitol. Applied Science Publishers, London, p. 1-9 (1978)). However, the purification of pure D-xylose is very expensive and pollutive step (see: Kind, V. B. et al., Gidroliz. Lesokhim. Promst., 3:11-12 (1987)). In addition, the hydrogenation of the D-xylose to xylitol at high temperature and high pressure using Raney-nickel metal catalyst is dangerous and pollutive (see: Hyvönen, L., et al., In Advances in Food Research, Vol. 28, eds Chishester, C. O., Mrak, E. M. and Stewart, G., Academic Press, New York, pp. 373-403 (1982)).
The existing drawbacks of conventional xylitol production methods including high pollution levels and waste-treatment concerns motivated researchers to develop alternative ways for its production. One of the most attractive procedures is biological production. The biological production does not require high purity of the substrate, D-xylose, and is a safe and environmentally-friendly process. Xylitol is produced by natural xylose-assimilating yeasts and fungi, such as Pachysolen tannophiulus, Candida guilliermondii, Candida parapsilosis, and Candida tropicalis (see: Dahiya, J. S., Can. J. Microbiol., 37:14-18 (1991), Yahashi, Y., et al., J. Ferment. Bioeng., 81:148-152 (1996), Kim, S. Y., et al., Food Sci. Biotechnol., 7:282-285 (1998), Morimoto, S., et al., J. Ferment. Technol., 64:219-225 (1986)).
Although Candida sp. was reported to be the most active and thus potentially most useful strain, the industrial production of xylitol has yet to be achieved because of the high production costs associated with the substrate, D-xylose, an expensive raw material with a low yield of xylitol. Efforts to develop more cost-effective methods of production have included using controlling the dissolved oxygen (see: Kim, S. Y., et al., U.S. Pat. No. 5,686,277 (1997)). However, the controlling the dissolved oxygen at the level of 0.8-1.2% is not easy in the industrial scale and, as a result, the xylitol yield was below 70% and productivity could not achieve an economical efficiency.
To solve these problems, the construction of a novel xylitol production strain with high-yield and high-productivity using metabolic engineering has been continuously required in the art.